Detection of circulating tumor cells during follow-up of patients with early breast cancer: Clinical utility for monitoring of therapy efficacy.

INTRODUCTION: Circulating tumor cells (CTCs) detection prior to and during therapy is considered as an independent and strong prognostic marker. The present study was designed to isolate and characterize CTCs in peripheral blood of an early breast cancer (BC) patient as a biomarker for monitoring treatments efficacy. MATERIALS AND METHODS: In total, 54 early breast cancer patients undergoing neoadjuvant and/or adjuvant chemotherapy regimens were enrolled into a prospective study. CTC detection in blood was performed by AdnaTest BreastCancer(™) (AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC-lysates. Additionally, cDNA from isolated CTCs and PBMC was used for qPCR gene expression analysis of TOP1, TOP2A, CTSD, ST6, CK19 and reference gene actin. RESULTS: We found that CTCs can be detected in the peripheral blood of approximately 31% of early stage breast cancer patients. The presence of CTCs was detected in 36% ER positive, 32% PR positive and 30% HER2 positive patients. We found no correlation between CTCs and tumor size, tumor grade, histological grade and receptor status. Only 7% of all patients remained CTCs positive after adjuvant therapy. Gene expression analysis revealed a particular heterogeneity of the studied genes. CONCLUSIONS: In conclusion, CTC detection may be a promising early marker of disease progression potentially enhancing the difficult therapeutic decisions. Further studies should, however, clearly demonstrate its utility for both the prediction of outcome and monitoring the effect of treatment.

Matrix metalloproteinases in serum and the follicular fluid of women treated by in vitro fertilization.

PURPOSE: To assess levels of matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) in follicular fluids and sera of female patients undergoing in vitro fertilization (IVF) treatment, and discover the role of MMPs in IVF outcome prediction. METHODS: Sera and follicular fluids were obtained from 58 female patients treated for infertility by IVF. Twenty-nine of them became pregnant after the embryo-transfer; another 29 were not successful in IVF and did not conceive. Forty female non-pregnant blood donors and 38 healthy pregnant patients in the first trimester after the physiological conception were examined as control groups. MMP-2 and MMP-9 were quantitively assessed using enzyme-linked immunosorbent assay. RESULTS: IVF females successfully conceiving after the IVF have shown the highest MMP-9 concentrations in sera (833.5 {686.0;958.7} ng/mL) and follicular fluids (9.6 {6.0; 17.0} ng/mL) compared to all other examined cohorts. Different behavior of MMP-2 and MMP-9 during the artificial ovulation was confirmed, because only MMP-9 has shown a vast difference between serum and follicular concentrations. CONCLUSIONS: MMP-9 could be a good predictor of the successful IVF outcome (pregnancy), which was proven for serum as well as follicular MMP-9 levels.

Plasma calprotectin in chronically dialyzed end-stage renal disease patients.

OBJECTIVE: The current study aimed to evaluate plasma calprotectin levels and clearance end-stage renal disease (ESRD) patients with and without acute infection undergoing chronic hemodialysis (HD). MATERIALS AND METHODS: Blood samples from 54 HD patients were obtained before and after the HD and 42 healthy blood donors were examined as controls. The blood levels of calprotectin, procalcitonin, C-reactive protein (CRP), and intracellular production of interleukins 10 and 12 in monocytes were determined in both groups. RESULTS: The concentrations of plasma calprotectin in ESRD patients were significantly higher than in healthy controls (p < 0.05). No differences between pre- and post-HD calprotectin plasma levels were observed (p = 0.07 for two-tailed test). Plasma calprotectin levels were not significantly influenced by the presence of acute infection (p = 0.19) or diabetes (p = 0.42). A significant positive correlation of plasma calprotectin to plasma beta-2 microglobulin was proven (p < 0.05). Procalcitonin (PCT), CRP, IL-10, and IL-12 were not correlated with plasma calprotectin before or after HD. The elevation of plasma calprotectin was correlated strongly to the hemodialysis vintage (r = 0.55, p < 0.01). CONCLUSIONS: Significantly elevated levels of plasma calprotectin in ESRD patients occur without an acute infectious cause and are not affected by the presence of diabetes. By analogy to plasma beta-2 microglobulin, a close relation of plasma calprotectin to HD vintage was shown.

Changes of immunological profiles in patients with chronic myeloid leukemia in the course of treatment.

In the previous paper of ours we compared, prior to start any treatment, a number of immunological parameters in 24 chronic myeloid leukemia patients with the same number of healthy subjects matched by age and sex. We found significant differences in the levels of immunoglobulins, the C4 component of complement, the C-reactive protein, interleukin 6, the composition of lymphocyte population and the production of some cytokines by stimulated CD3+ cells. Eleven of these patients were followed longitudinally. After treatment with hydroxyurea, interferon alpha, imatinib mesylate and dasatinib, or various combinations thereof, hematological remission was achieved in all patients and complete cytogenetic remission in nine of them. There was a nearly general tendency towards normalization of the abnormalities observed in the patients at their enrollment.

Concentrations of sRAGE in serum and follicular fluid in assisted reproductive cycles--a preliminary study.

BACKGROUND: To investigate inflammatory processes during ovarian hyperstimulation, we have studied the soluble receptor for advanced glycation end products (sRAGE) levels in sera and follicular fluids of women undergoing in vitro fertilisation (IVF) cycle. METHODS: A total of thirty-three women undergoing IVF treatment were recruited, the number of follicles in investigated IVF cycles was 18 +/- 10 (mean +/- SD), oocytes 12 +/- 8, and the clinical pregnancy rate was 42% (14/33). The control group of serum samples was comprised of 35 healthy female blood donors. Sera and follicular fluids were examined for sRAGE levels by enzyme-linked immunosorbent assay (sRAGE ELISA, Quantikine, R&D Systems). RESULTS: Serum levels of sRAGE in women after ovarian hyperstimulation and induction of ovulation (1039 +/- 493 pg/mL) were significantly lower than in healthy blood donors (1535 +/- 438 pg/mL), p = 0.045. Follicular sRAGE levels (4355 +/- 1100 pg/mL) were significantly higher than serum levels (1039 +/- 493 pg/ml), p < 0.001. Serum sRAGE levels showed significant negative correlation with the number of stimulated follicles (r = -0.71, p = 0.01) and retrieved oocytes (r = -0.54, p = 0.048). Women who successfully conceived after the IVF showed significantly higher sRAGE levels in follicular fluid (4595 +/- 925 pg/mL) compared to women who did not conceive (3986 +/- 806 pg/mL), p = 0.031. CONCLUSIONS: Concentration of sRAGE in follicular fluid is several-fold higher compared to serum and most other biological fluids investigated until this time. It supports the hypothesis that mammalian ovulation can be compared to an inflammatory event. A significant negative correlation of serum sRAGE with the yield of follicles and oocytes, together with the high follicular sRAGE levels, in particular in women who conceive after the IVF, could be explained by the essential outflow of sRAGE to the follicular compartment.

Anti-inflammatory effect of biological treatment in patients with inflammatory bowel diseases: calprotectin and IL-6 changes do not correspond to sRAGE changes.

AIM: The main objective was to examine the relationship between the soluble receptor for advanced glycation end products (sRAGE) and calprotectin concentrations in faeces and serum of patients with inflammatory bowel diseases (IBD) during biological treatment with infliximab. MATERIALS AND METHODS: A total of 29 IBD patients treated with infliximab were evaluated. Calprotectin and sRAGE in serum and faeces and serum IL-6 and CRP were measured during the induction regimen of infliximab treatment at weeks (W) 0, 2 and 10. RESULTS: At W0, a significant increase in faecal calprotectin was found in IBD compared to healthy persons (690 +/- 696 microg/g and 23 +/- 7 microg/g, respectively, p < 0.001). No clear difference was found in serum sRAGE levels in IBD cohort compared to healthy controls (772 +/- 274 pg/mL and 720 +/- 107 pg/mL, respectively, p = 0.159); however, a significant negative correlation was found between faecal calprotectin levels and serum concentrations of sRAGE in the active IBD cohort (r = -0.518, p = 0.004). In the stool eluates, sRAGE levels were non-measurable. In the group of responders-to-treatment, the initial surge in both faecal and serum calprotectin levels as well as CRP and IL-6 was followed by a significant decrease on W10. Surprisingly, no significant changeovers were seen in serum sRAGE concentrations in responders neither in W2 nor in W10. CONCLUSIONS: Unlike other examined local and systemic inflammatory markers, serum sRAGE did not change during the infliximab treatment, despite the initial correlation with the degree of mucosal inflammation.

Detection of galectin-3 in patients with inflammatory bowel diseases: new serum marker of active forms of IBD?

OBJECTIVE: It is an open question whether multifunctional galectin-3 can be a serum marker in inflammatory bowel disease. METHODS: Western blots and commercial ELISA detected and quantitated the lectin immunocytochemistry using double labeling localized it in tissue sections. RESULTS: Serum concentrations were significantly increased in specimen of patients with active and remission-stage ulcerative colitis and Crohn's disease, associated with emerging positivity of CD14(+) cells. CONCLUSION: Enhanced concentration of galectin-3 in serum reflects presence of disease and points to its involvement in the pathogenesis.

Novel anti-carbohydrate autoantibodies in patients with inflammatory bowel disease: are they useful for clinical practice?

The objective of this study was to test the diagnostic accuracy of novel anti-carbohydrate assays in patients with inflammatory bowel disease, namely in Crohn's disease. These carbohydrate assays are based on oligosaccharide chitobioside carbohydrate - anti-chitobioside carbohydrate antibodies (ACCA), laminaribioside carbohydrate anti-laminaribioside carbohydrate antibodies (ALCA), and mannobioside carbohydrate - anti-mannobioside carbohydrate antibodies (AMCA). We compared these assays with the anti-Saccharomyces cerevisiae antibodies (ASCA) assay. The results of this study suggest that ASCA are still the best serological marker for Crohn's disease. Further studies are required to explore the clinical utility of ACCA, ALCA and AMCA.

Identification of rice proteins recognized by the IgE antibodies of patients with food allergies.

Similarity among food allergens is a great problem affecting the specificity of diagnosis and treatment of allergic patients. We have observed that 80% of patients with food (including wheat) and pollen allergies have increased IgE antibodies against rice proteins. By immunoblotting, we documented that boiling decreased solubility and IgE reactivity of PBS-extracted rice and wheat proteins, yet in SDS extracts this reactivity was only slightly changed. The sera of patients highly positive on the IgE immunoblot and positive in basophil activation and skin prick test with boiled rice components were used for characterizing the IgE-binding proteins separated by 1D or 2D electrophoresis. Using mass spectrometry, we identified 22 rice SDS soluble proteins. Six of them were new thermostable potential rice allergens: glutelin C precursor, granule-bound starch synthase 1 protein, disulfide isomerase-like 1-1 protein, hypothetical protein OsI_13867, putative acid phosphatase precursor 1, and a protein encoded by locus Os02g0453600. All of the identified rice proteins differed from known wheat allergens, except proteins belonging to the α-amylase/trypsin inhibitor family. Furthermore, we would suggest that in patients with high IgE reactivity to wheat and rice components, the IgE immunoblot and skin prick test with boiled rice proteins could be beneficial before diet recommendation.

Phosphatidylserine-dependent anti-prothrombin antibodies (aPS/PT) in infliximab-treated patients with inflammatory bowel diseases.

PURPOSE: To (1) examine the occurrence and concentrations of aPS/PT and aPL in inflammatory bowel disease (IBD) patients at the beginning of and during anti-TNF-alpha therapy with infliximab; (2) investigate the link of the aPS/PT and aPL presence with antibodies to infliximab (ATI) formation; and (3) examine possible clinical consequences of aPS/PT and/or aPL positivity in IBD patients. MATERIALS AND METHODS: Thirty (30) IBD patients treated with infliximab were analyzed regarding aPS/PT, aPL, and ATI antibody serum levels by standardized ELISAs at treatment weeks 2 (W2) and 14 (W14). RESULTS: At W2, 40 % of infliximab-treated patients had elevated aPS/PT and 16.7 % had elevated aPL serum levels. At W14, the proportion of aPS/PT-positive sera decreased to 16.6 %, whereas aPL distribution remained unchanged. Moreover, concentrations of aPS/PT have shown significant differences at W2 (16.64 [10.06; 33.06] U for IgG and 18.46 [9.18; 32.48] U for IgM) and at W14 (8.24 [2.78; 19.82] U for IgG and 8.57 [5.55; 26.82] U for IgM), p = 0.009 and p = 0.003, respectively. In ATI-positive samples, aPS/PT IgG were more frequent (p = 0.001 for W2 and p = 0.003 for W14), whereas aPS/PT IgM and aPL IgG/IgM did not show such association. CONCLUSIONS: Higher concentrations of aPS/PT IgG and IgM were found in IBD patients at the beginning of the biological treatment period compared to the maintenance treatment period. Moreover, aPS/PT IgG were more frequent in ATI-positive individuals, which was not observed in aPL. We speculate that there is a relationship between the aPS/PT and the severity of inflammation and auto-aggressive processes in IBD.

Intracellular cytokine production in peripheral blood lymphocytes: a comparison of values in infertile and fertile women.

PROBLEM: To analyze the relation of the fertility and pregnancy of women of childbearing age to the intracellular (IC) production of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and interleukins 2 and 4 (IL-2 and IL-4). METHOD OF STUDY: Intracellular cytokine production in peripheral blood CD3(+) CD4(+) lymphocytes was analyzed by flow cytometry in 185 women being treated for infertility and 50 fertile women of childbearing age. RESULTS: Infertile women have a significantly higher IC production of TNF-α, IFN-γ, IL-2, and IL-4 and higher ratios of TNF-α/IL-2, TNF-α/IL-4, and TNF-α/IFN-γ compared to the fertile women. CONCLUSION: Cytokines produced by Th lymphocytes are important in orchestrating the immune response during conception, and Th-cell dysregulation could be a reason for infertility.

Impaired deoxyribonuclease I activity in patients with inflammatory bowel diseases.

Background and Aims. Deoxyribonuclease I (DNaseI) is an endonuclease that facilitates chromatin breakdown and promotes susceptibility to autoimmune disorders. The aim of current study was to investigate serum DNase I activity in patients with inflammatory bowel diseases (IBD). Patients and Methods. A cohort of 110 IBD patients was evaluated, aged 35 ± 12 years, 77 with Crohn's disease (CD) and 33 with ulcerative colitis (UC). 50 SLE patients and 50 healthy blood donors were examined as control groups. Results. DNase I activity in IBD patients was significantly lower than in healthy individuals, but higher than in SLE patients (P < .0001). Patients with UC showed higher DNase I activity than CD patients, P = .21. DNase I activity in female patients with IBD was significantly lower than in males, P = .024; however, no differences in DNase I activity were found in relation to gender in healthy individuals. DNase I activity has shown a strong negative correlation with the serum concentration of anti-nucleosomal antibodies in the autoimmune (SLE + IBD) cohort, as well as in the separate IBD cohort. Conclusions. Reduced serum DNase I activity probably has pathogenetic consequences in IBD. Induction of autoantibodies towards nucleosomes could be a reflection of impaired DNase I activity.

Anticarbohydrate antibodies as markers of inflammatory bowel disease in a Central European cohort.

BACKGROUND: The study discusses the role of antichitobioside carbohydrate antibody (ACCA), antilaminaribioside carbohydrate antibodies (ALCA), and antimannobioside carbohydrate antibodies (AMCA) in Central European patients with inflammatory bowel disease (IBD). PATIENTS AND METHODS: Twohundred and seventy-two serum samples were used - 116 Crohn's disease (CD), 84 ulcerative colitis, and 72 healthy control samples. All samples were evaluated using enzyme-linked immunosorbent assay for the following four anticarbohydrate assays: ACCA, ALCA, AMCA, and anti-Saccharomyces cerevisiae antibodies (gASCA). RESULTS: gASCA antibodies showed the highest sensitivity (67%) for a CD diagnosis, followed by AMCA (31%), ACCA (27%), and ALCA (25%). Positivity of at least one of the four assays increased the overall sensitivity of antibody testing in CD up to 85.5%. Mean serum gASCA levels were significantly higher in CD patients who were younger at diagnosis and had a longer disease duration before blood sampling (P<0.001). In nonstricturing, nonpenetrating CD, serum gASCA levels were lower than in patients with stricturing and/or penetrating behavior (P<0.05). The strongest association of gASCA was found with ileocolonic CD and with upper gastrointestinal disease (P<0.001). No association between anticarbohydrate (AMCA, ACCA, and ALCA) antibodies and CD location, behavior, age at onset, and disease duration was found; however, that sample size of some of our subgroups was probably too small to make firm conclusions on associations with all CD phenotypes. None of the assessed anticarbohydrate assays was predictive of colonic CD in patients in whom the distinction between CD and ulcerative colitis is not obvious using routine diagnostic methods. There was no relationship between the presence or concentration of anticarbohydrate antibodies and the inflammation measured by C-reactive protein levels. CONCLUSION: The use of a panel of anticarbohydrate antibodies may provide additional help in distinguishing IBD from non-IBD disease patterns. The addition of AMCA, ALCA, and ACCA assays as IBD serology markers improves the overall sensitivity of immunological examinations in IBD; however, anticarbohydrate assays are not helpful for predicting CD behavior.